When mammalian cell cultures, such as mouse embryo cells or human fibroblasts, are maintained in vitro by conventional techniques in which the basal nutrient medium is supplemented with serum, the cultures undergo growth crisis or senescence. During this period the normal cells of the cultures gradually cease proliferation and eventually degenerate. In some cases this phenomenon is followed by the outgrowth of genetically abnormal cells capable of indefinite growth in culture. It has been suggested that crisis or senescence in vitro may be related to aspects of aging in the intact organism. It is reasonable to consider that the serum supplement to the culture media may exert negative as well as positive influences on cell proliferation, and negative effects of serum factors might contribute to the failure of genetically normal cells to proliferate under conventional conditions. We developed a serum-free medium for mouse embryo cells and found that cultures established under these conditions did not undergo crisis and maintained a normal karyotype. Serum-free derived mouse embryo cells were markedly growth-inhibited by serum or plasma; this inhibition was reversible upon removal of serum from the culture medium. We propose three types of studies as extensions of these observations: (I) application of these techniques to human fibroblasts in culture, in an attempt to determine the degree to which senescence of human cells in vitro may be influenced by the extracellular environment; (II) characterization of the inhibitory factor(s) in plasma using an assay designed to differentiate inhibitory activities of physiological significance from nonphysiological toxicities that may be encountered as artifacts; (III) cell fusion studies aimed at an examination of the mechanism of growth inhibition by serum and the relationship of this effect to the phenomenon of senescence in vitro.